Structural and Biochemical Analyses of Mycobacterium tuberculosis N-Acetylmuramyl-l-alanine Amidase Rv3717 Point to a Role in Peptidoglycan Fragment Recycling

Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial N-acetylmuramyl-l-alanine amidase, Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared with previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded β-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn²⁺ binding rearranges active site residues, and disulfide formation promotes folding of the β-hairpin. Although Zn²⁺ is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (Glu-200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling.     

Fossil insects of the middle and upper Permian of European Russia

Fossil insects of European Russia from the Urzhumian to Vyatkian stages are reviewed, new taxa are described, and dynamics of insect taxonomic diversity around the Permian-Triassic boundary in light of the Paleozoic-Mesozoic boundary global extinction problem is analyzed. Traces of interactions between arthropods and plants are analyzed. Insect-bearing deposits of the Late Paleozoic found in the northern and eastern areas of the East European Platform are unique on the global scale in their completeness and continuity, allowing us to trace especially comprehensively the biotic processes that occurred around the boundary described as the time of the greatest biotic catastrophe of the Phanerozoic. A total of 28 genera and 111 species are newly described. Within the range from the Urzhumian to the Permo-Triassic boundary, 15 representative successive assemblages, including 112 families, are recognized (seven in the area in question and eight in other regions of Asia, Australia, and Africa). New tools are developed for the analysis of the dynamics of diversity. These tools show an approximately equilibrium (slightly positive) dynamics in the Urzhumian and Severodvinian and a drop in diversity during the Vyatkian Age. It is shown that Permian insect assemblages acquired a substantially post-Paleozoic pattern much earlier than the end of the Paleozoic. The character of changes that took place in the Induan and Olenekian remains uncertain, but a large-scale extinction event did not occur here: most families that have not been recorded at the beginning of the Triassic are recorded again in the Middle and Upper Triassic. Nevertheless, a biotic crisis probably actually took place, but was reduced to reorganization of the biota’s structure, which provided enormous growth of biodiversity over subsequent hundreds of millions of years, rather than resulted in catastrophic extinction. This study is intended for entomologists, stratigraphers, and all readers interested in the biotic events that took place around the Permian-Triassic boundary.     

New and little-known Eoblattida (Insecta) from the Paleozoic of Russia

New taxa of the insect order Eoblattida from the Upper Carboniferous of Russia are described, including Narkemina kata sp. nov., Narkeminopsis inversa sp. nov., Carbonokata storozhenkoi gen. et sp. nov., Tshunoptera ampla gen. et sp. nov., and Evenkiophlebia collucata gen. et sp. nov. from the Chunya locality (Krasnoyarsk Region), Narkemulla sibirica gen. et sp. nov. from the Chunya and Izykhskie Kopi localities, Khakassia (all Cnemidolestidae), and Izykhia tridentis gen. et sp. nov. from Izykhskie Kopi (?Spanioderidae). Narkeminuta permiana gen. et sp. nov. (Cnemidolestidae) is described from the Kedrovka locality (Kemerovo Region; Lower Permian), and Issadische maximum gen. et sp. nov. (Eoblattida incertae familiae) is descriobed from the Isady locality (Vologda Region; Upper Permian). Permeoblatta borealis Rasnitsyn et Aristov, 2010 (Idelinellidae) from Isady is redescribed.     

Polymorphisms and haplotypes in MyD88 are associated with the development of sarcoidosis: a candidate-gene association study

Sarcoidosis is considered as a disorder of protracted immune response to an as yet unidentified causative agent that leads to granuloma formation. Material from M. tuberculosis and P. acne has been repeatedly detected in the sarcoidosis lesions, implying the involvement of the Toll-like receptor2 (TLR2) gene that responds to these intracellular pathogens. Since TLR2 association studies have produced controversial results, we sought to investigate whether the downstream signalling molecule MyD88 could be linked to disease susceptibility. We analyzed a total of 93 cases with sarcoidosis and of 89 controls for the most common MyD88 SNPs: ?938C>A (rs4988453) and 1944C>G (rs4988457). There is evidence that the genotype distributions of both variants are associated with the development of sarcoidosis (p = 0.038 for ?938C>A and p = 0.026 for 1944C>G). In particular, ?938A and 1944G carriers were associated with risk of sarcoidosis [OR = 2.48 (1.23–5.02) and OR = 0.33 (0.14–0.76)], respectively, indicating dominance of the mutant alleles; however, the adjustment of the effect size for age and sex diminished the significance. The haplotype analysis showed association for the ?938A/1944G haplotype (p < 0.001). Since genetic association studies have linked MyD88 to Hodgkin’s lymphoma it is tempting to speculate that MyD88 may contribute to the granuloma formation that characterizes sarcoidosis.     

Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69–99% of control by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.     

Revision of the family Epideigmatidae (Insecta: Grylloblattida)

The family Epideigmatidae is revised. New members of this family from the Lower Permian of Russia: Vilvaptera permyakovae, gen. et sp. nov. (Vilva locality, Artinskian Stage, Perm Region) and Tshekardeigma rasnitsyni, gen. et sp. nov. (Chekarda locality, Kungurian Stage, Perm Region) are described. Paraphenopterum unicolor Storozhenko, 1992 (Soyana locality, Kazanian Stage, Arkhangelsk Region) is redescribed. The family Stenoneuritidae is regarded as a synonym of Epideigmatidae; the genera Fayoliella Meunier, 1908 (Upper Carboniferous of France), Fabreciella Carpenter, 1934 (Upper Carboniferous of the USA), and Turbopterum Kukalová, 1964 (Lower Permian of the Czech Republic) are transferred to Epideigmatidae. Fabreciella allegheniensis Carpenter, 1934 is synonimized under F. pennsylvanica Carpenter, 1934.     

Photoacoustic and fluorescent effects in multilayer plasmon‐dye interfaces

Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near‐infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold‐ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low‐toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near‐infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.      

The DYW-class PPR protein MEF7 is required for RNA editing at four sites in mitochondria of Arabidopsis thaliana

In plant mitochondria and plastids, RNA editing alters about 400 and about 35 C nucleotides into Us, respectively. Four of these RNA editing events in plant mitochondria specifically require the PPR protein MEF7, characterized by E?and DYW extension domains. The gene for MEF7 was identified by genomic mapping of the locus mutated in plants from EMS treated seeds. The SNaPshot screen of the mutant plant population identified two independent EMS mutants with the same editing defects as a corresponding T-DNA insertion line of the MEF7 gene. Although the amino acid codons introduced by the editing events are conserved throughout flowering plants, even the combined failure of four editing events does not impair the growth efficiency of the mutant plants. Five nucleotides are conserved between the four affected editing sites, but are not sufficient for specific recognition by MEF7 since they are also present at three other sites which are unaffected in the mutants.     

Multiple specificity recognition motifs enhance plant mitochondrial RNA editing in vitro

Analysis of RNA editing in plant mitochondria has at least in vitro been hampered by very low activity. Consequently, none of the trans-acting factors involved has yet been identified. We here report that in vitro RNA editing increases dramatically when additional cognate recognition motifs are introduced into the template RNA molecule. Substrate RNAs with tandemly repeated recognition elements enhance in vitro RNA editing from 2-3% to 50-80%. The stimulation is not influenced by the editing status of a respective RNA editing site, suggesting that specific recognition of a site can be independent of the edited nucleotide itself. In vivo, attachment of the editing complex may thus be analogously initiated at sequence similarities in the vicinity of bona fide editing sites. This cis-acting enhancement decreases with increasing distance between the duplicated specificity signals; a cooperative effect is detectable up to approximately 200 nucleotides. Such repeated template constructs promise to be powerful tools for the RNA affinity identification of the as yet unknown trans-factors of plant mitochondrial RNA editing.